Reactive nanocomposites and methods of making the same

ABSTRACT

Reactive nanocomposites comprising a metal nanoparticle functionalized with one or more layers of self-assembled protein cages and methods of making the same. The reactive nanocomposites according to the present invention demonstrate improved reaction kinetics and enhanced exothermic behavior.

RIGHTS OF THE GOVERNMENT

The invention described herein may be manufactured and used by or forthe Government of the United States for all governmental purposeswithout the payment of any royalty.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of energeticnanomaterials. More particularly, it relates to reactive nanocompositescomprising functionalized metal nanoparticles and methods of making thesame.

2. Description of the Related Art

Proteins such as ferritin that form protein cages have been used in avariety of nanocomposite materials. For example, protein cages have beenused for the confined synthesis of monodisperse Au, Ag, CdS, Pd, TiO₂,Fe₃O₄, and UO₂ nanoparticles by adding a nanoparticle-binding peptide.In addition, protein cages have been used in the moleculartransport/storage of small molecules for drug delivery and for assemblyon titania, carbon nanotube, and gold surfaces by addition of ametal-binding peptide on the exterior cage. In these assemblies, theclose proximity of ferritin to the nanomaterial surface resulted inenhanced optical and electrical properties.

The use of nanoparticles to fabricate reactive nanocomposites combinesthe high reaction rates of molecular explosives and materials with thehigh energy density of composite materials. However, the performance ofmany nanomaterials-based energetic formulations often suffers from poormass transport, uneven distribution of nanocomposite components, andlarge diffusion distances. In addition, conventional methods ofmanufacturing reactive nanocomposites often encounter a number ofproblems associated with safe handling and processing of the materialssuch as their propensity toward decomposition and instability andsensitivity of the reactive components. Several methods such as powdercompaction, melt blending, and solution mixing that attempt to achieve auniform nanocomposite have met with limited success and are stillplagued with poor chemical and physical interaction between thecomponents of the nanocomposite.

SUMMARY OF THE INVENTION

The present invention includes a method of making a reactivenanocomposite comprising the steps of: loading positively-chargedprotein cages with one or more types of oxidizer to form loadedpositively-charged protein cages, with the oxidizer being loaded intothe pores of the positively-charged protein cages; and assembling theloaded positively-charged protein cages onto an outer surface of metalnanoparticles to form the reactive nanocomposite.

In one embodiment of the method, the metal nanoparticles are Al, B, Si,Mg, Ni, Ti, and/or Ag. In another embodiment, the oxidizer is iron oxideand/or ammonium perchlorate. In a further embodiment, thepositively-charged protein cages are ferritin, heat shock proteins,capsid proteins, and/or ferritin-like proteins. In an alternativeembodiment, the positively-charged protein cages further comprise achemical oxidizing agent, a non-ferric metal oxide, a molecularexplosive, and/or a fluorescent dye taggant.

The present invention further includes a method of making a multi-layerreactive nanocomposite comprising alternating layers ofoppositely-charged loaded protein cages. The method comprises the stepsof: loading positively-charged protein cages with one or more types ofoxidizer to form loaded positively-charged protein cages, with theoxidizer being loaded into the pores of the positively-charged proteincages; assembling the loaded positively-charged protein cages onto anouter surface of metal nanoparticles to form a one-layer reactivenanocomposite; loading negatively-charged protein cages with one or moretypes of oxidizer to form loaded negatively-charged protein cages, withthe oxidizer being loaded into the pores of the negatively-chargedprotein cages; assembling the loaded negatively-charged protein cagesonto the one-layer reactive nanocomposite to form a two-layer reactivenanocomposite; and adding alternating layers of loadedpositively-charged protein cages and loaded negatively-charged proteincages to achieve the multi-layer reactive nanocomposite having a desiredcomposition and a desired number of layers.

In one embodiment of the method, the metal nanoparticles are Al, B, Si,Mg, Ni, Ti, and/or Ag. In another embodiment, the oxidizer is iron oxideand/or ammonium perchlorate. In a further embodiment, thepositively-charged protein cages are ferritin, heat shock proteins,capsid proteins, and/or ferritin-like proteins. In an alternativeembodiment, the positively-charged protein cages further comprise achemical oxidizing agent, a non-ferric metal oxide, a molecularexplosive, and/or a fluorescent dye taggant.

The present invention further includes a method of making a multi-layerhybrid reactive nanocomposite comprising alternating layers of at leastone of oppositely-charged loaded protein cages and negatively-chargedpolyelectrolyte complexes. The method comprises the steps of: loadingpositively-charged protein cages with one or more types of oxidizer toform loaded positively-charged protein cages, with the oxidizer beingloaded into the pores of the positively-charged protein cages;assembling the loaded positively-charged protein cages onto an outersurface of metal nanoparticles to form a one-layer reactivenanocomposite; coating at least one type of oxidizer with anegatively-charged polyelectrolytes to form negatively-chargedpolyelectrolyte complexes; assembling the negatively-chargedpolyelectrolyte complexes onto the one-layer reactive nanocomposite toform a two-layer hybrid reactive nanocomposite; and adding alternatinglayers of at least one of loaded positively-charged protein cages,loaded negatively-charged protein cages, and negatively-chargedpolyelectrolyte complexes to achieve a multi-layer hybrid reactivenanocomposite having a desired composition and a desired number oflayers.

In one embodiment of the method, the metal nanoparticles are Al, B, Si,Mg, Ni, Ti, and/or Ag. In another embodiment, the oxidizer is iron oxideand/or ammonium perchlorate.

In an alternative embodiment, the method of making a multi-layer hybridreactive nanocomposite further comprises site-directed assemblycomprising the steps of: coating a surface with negatively-chargedpolyelectrolytes, in which the surface has a desired location; andassembling the one-layer reactive nanocomposite onto thenegatively-charged polyelectrolytes, thereby directing assembly of themulti-layer hybrid reactive nanocomposite onto the surface in thedesired location.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a depiction of one embodiment of a single-layer reactivenanocomposite.

FIG. 1B is a transmission electron microscope (TEM) image of cationizedferritin protein cages filled with an iron oxide core that wereassembled onto nano-Al particles.

FIG. 2A is a depiction of one embodiment of a multi-layer reactivenanocomposite.

FIG. 2B is a depiction of an alternative embodiment of a multi-layerreactive nanocomposite.

FIGS. 3A-3F are TEM images of single- and multi-layer reactivenanocomposites respectively containing between one and six alternatinghomogeneous layers of cationized and native ferritin loaded with ironoxide assembled onto nano-Al particles.

FIG. 4 is a quartz crystal microbalance plot of reactive nanocompositescomprising between one and six layers of iron oxide-loaded ferritins ofequal mass.

FIG. 5 is a dynamic light scattering plot of reactive nanocompositescomprising between one and six layers of iron oxide-loaded ferritins ofequal mass.

FIG. 6 is the thermogravimetric analysis (TGA)/differential thermalanalysis (DTA) profile of bio-thermite and unfunctionalized nano-Al.

FIG. 7 is the TGA/DTA profile of AP-loaded ferritin-nano-Al andunfunctionalized nano-Al.

FIG. 8 is the TGA/DTA profile of unfunctionalized nano-Al andmulti-layer ferritin-nano-Al containing one to four homogeneous layersof iron oxide-loaded ferritin.

FIG. 9 is the TGA/DTA profile for four-layer ferritin-nano-Al plottedwith the TGA/DTA profile generated from a bulk thermite reaction ofmicron- or nano-sized iron oxide particles and nano-Al particles.

FIGS. 10A-D show the combustion characteristics of several reactivenanocomposites utilizing a high speed camera.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes reactive nanocomposites comprising ananoparticle functionalized with one or more layers of self-assembledproteins and/or protein cages and methods of making the same. Thepresent invention takes advantage of assembly strategies derived frombiology and the high affinity of biomolecules for inorganic materials todirect the layer-by-layer (LBL) assembly of oxidizer-loaded proteincages onto the surface of metal nanoparticles. The resulting reactivenanocomposites demonstrate improved reaction kinetics, due in part tothe reduction in the diffusion distance between the reactants and theincreased stability of the oxidizing agent inside the protein cage. Theability to tailor the number and composition of the protein layers maybe used to control and optimize stoichiometric conditions, therebytuning and maximizing energetic performance. The result is astoichiometrically balanced energetic reaction in which substantiallyall of the reactive metal may be consumed. The reactive nanocompositesaccording to the present invention demonstrate enhanced exothermicbehavior in comparison to other reactive materials such as nanothermitemixtures of bulk nano-Al with free ammonium perchlorate and/or micron-and nano-sized iron oxide powders prepared without the use of ferritin.

In one embodiment of the present invention, an improved reactivenanocomposite may be fabricated by loading protein cages with anoxidizer and assembling one or more layers of the loaded protein cagesonto the surface of a reactive metal nanoparticle. The nanometal may beany suitable energetic material including Al, B, Si, Mg, Ni, Ti, and Ag,with Al being one of the most common. The assembled nanocomposite maycomprise one type of metal or a combination of metals to create aheterogeneous complex. Reactive metals contain and release a largeamount of stored energy due to their chemical composition and size. As aresult, they are regularly used in propellants, explosives, andpyrotechnics. Many conventional formulations utilize micron-scalereactive metal powders as fuel and/or additives to achieve and improvecombustion efficiency and energy output. Alternatively, metal preparedas a nanopowder such as nano-aluminum (nano-Al) is of particularinterest as an energetic material because of its superior properties andprocessability. Nanometals generally have a higher energy density thanorganic explosives and a higher specific surface area as compared tomicron-sized metal powders. In addition, energetic nanometals areamenable to functionalization and assembly with other reactivematerials, and they often contain a thin oxide layer, which makes themless pyrophoric. All of these properties contribute to the potential forenhanced rates of reaction for composites comprising nanometals.

A protein cage comprises multiple protein units that self assemble intoa cage surrounding a central cavity. Ferritins are an important familyof highly-conserved, globular proteins that regulate in vivo ironlevels, and they one of the most commonly used protein cages. They aretypically about 12 nm in diameter, with an internal cavity diameter ofabout 8 nm Ferritins store reservoirs of Fe³⁺ as a ferrihydritenanoparticle, FeO(OH), within the hollow protein cavity, releasing Feions when needed. The protein is an abundant and inexpensive materialthat may be obtained recombinantly in high yields or from large ferritinreservoirs found in mammals (i.e. horse spleen).

Ferritin may be modified to improve the binding interaction with thenanometal particle. Three types of ferriting—a chemically modifiedferritin cage with a positively charged surface (cationized ferritin), anegatively charged unmodified native ferritin with a carboxylate-richsurface, and a genetically modified protein cage displaying Al-bindingpeptides—were tested to determine which demonstrated the best binding tonano-Al (data not shown). The cationized ferritin cages showed thehighest binding to nano-Al particles, while the genetically modifiedferritin cages showed about three-fold less binding to the aluminasurface than cationized ferritin by mass. The unmodified native ferritinshowed almost no affinity for the nano-Al.

Additional examples of self-assembling proteins that may be used includeheat shock proteins, capsid proteins derived from viruses andbacteriophages, and ferritin-like proteins, all of which possess diversestructures, geometries, sizes, and internal cavities (pores). Examplesof heat shock proteins may include TF55β (chimeric) and the smallheat-shock protein isolated from Methanococcus jannaschii. Examples ofviruses containing suitable capsid proteins may include the tobaccomosaic virus, brome mosaic virus, iridovirus, the cowpea mosaic virus,and the cowpea chlorotic mottle virus. Suitable bacteriophage capsidproteins may be derived from the MS2, M13, and P22 bacteriophages. Ingeneral, viral and bacteriophage capsid proteins tend to be larger,which allows them to encapsulate larger oxidizer molecules and/oradditional reactants. Many assembled viral capsids are around 30 nm indiameter, with some reaching 100 nm or more in diameter. Ferritin-likeproteins may include Dps proteins (DNA-binding proteins from starvedcells) from Escherichia coli and an iron-binding protein Dpr fromStreptococcus pyogenes.

The interior and/or exterior surfaces of the ferritin or other type ofprotein cage may be further modified by genetic or chemical addition ofmolecular recognition elements such as peptides, DNA aptamers, andantibodies, as well as fluorophores and polymers. Examples may includethe addition of a titanium-binding peptide to the exterior ferritinsurface, addition of silver-mineralizing peptides displayed along theinterior surface of the protein cage, and introduction of polymericdendrimer scaffolds of varying sizes synthetically grown from interiorsurface of a P22 capsid through a radical initiated polymerizationmechanism. These modified proteins may be used for a variety ofapplications, including sensing, binding, tracking, and imaging ofvarious targets such as bacteria, viruses, and chemical warfare agents.In addition, the modified protein cages may be used in the location andneutralization or destruction of targets upon binding and sequestrationof the target.

One example of a suitable oxidizer is NH₄ClO₄ (ammonium perchlorate,AP), a strong oxidizer that decomposes at low temperatures (<200° C.)and releases energy when mixed with reactive metals such as nano-Al.Another example of a suitable oxidizer is an iron oxide such asferrihydrite (FeO(OH)), as well different crystalline phases of ironoxide such as ferric oxide or hematite (Fe₂O₃) and ferrous ferric oxideor magnetite (Fe₃O₄), all of which are herein generally referred to as“iron oxide.”

In addition to the oxidizer, the protein cages may optionally contain avariety of additional reactants such as other types of chemicaloxidizing agents and non-ferric metal oxides, molecular explosives, andfluorescent dye taggants, and combinations thereof. Examples of chemicaloxidizing agents and metal oxides may include Ag(IO₃) (silver iodate),manganese oxide, copper oxide, and boron oxide. Examples of molecularexplosives may include trinitrotoluene (TNT) andcyclotrimethylenetrinitramine (RDX). Examples of taggants may includecadmium telluride (CdTe) or cadmium selenide (CdSe) quantum dots andrhodamine fluorescent dyes.

The reactive nanocomposite may optionally comprise one or more polymericelectrolytes (polyelectrolytes). Examples of suitable polyelectrolytesmay include, but are not limited to, poly-L-lysine (PLL), polyacrylicacid (PAA), poly(sodium styrene sulfonate) (PSS), poly(allylaminehydrochloride) (PAH), deoxyribonucleic acid (DNA), ribonucleic acid(RNA), and combinations thereof. Polyelectrolytes may be used in placeof or in addition to the charged protein cages to create abrick-and-mortar structure or to assist in the directed self-assembly ofan energetic formulation. For example, iron oxide coated with anegatively charged polyelectrolyte may be assembled onto nano-Alfunctionalized with a layer of cationized ferritin. In addition,polyelectrolytes may be used in site-directed assembly, for example, bycoating a surface with a negatively charged polyelectrolyte andassembling nano-Al functionalized with a layer of cationized ferritinonto the surface.

Referring now to the drawings, like reference numerals may designatelike or corresponding parts throughout the several views. In oneembodiment, the reactive nanocomposite may comprise a single layer ofprotein cages, an example of which is shown in FIG. 1A. Cationizedferritin cages 100 are loaded with an oxidizer, which may be an ironoxide nanoparticle 110 or AP 120, to form an iron oxide-loadedcationized ferritin cage 130 or an AP-loaded cationized ferritin cage140, respectively. The iron oxide nanoparticles 110 may be in thebiologically active form of ferrihydrite, FeO(OH). The iron oxide-loadedcationized ferritin cages 130 or the AP-loaded cationized ferritin cages140 are then assembled in a single layer onto the outer surface ofnano-Al particles 150 to form a single-layer reactive nanocomposite 160a, 160 b (see also Example 1). The single-layer reactive nanocomposite160 a comprising iron oxide-loaded cationized ferritin cages 130 ischemically equivalent to thermite (a metal oxide plus a reactive metal)and may be deemed a “bio-thermite.”

FIG. 1B is a transmission electron microscope (TEM) image of cationizedferritin protein cages filled with an iron oxide core that wereassembled with nano-Al particles (approximately 80 nm) to create abio-thermite such as the embodiment of a single-layer reactivenanocomposite 160 a depicted in FIG. 1A. The pore of each ferritin cagecontains a particle that is approximately 6 nm in diameter and is in thebiologically active form of ferrihydrite, FeO(OH), as depicted by thedark electron dense cores on the surface of the nano-Al particles in theTEM micrograph. As seen in FIG. 1B, the surface of each nano-Al particleis uniformly decorated with approximately 30-40 ferritin cages.

In an alternative embodiment shown in FIGS. 2A and 2B, the reactivenanocomposite according to the present invention may be a multi-layerreactive nanocomposite comprising two or more layers of different typesof protein and/or protein cages achieved by a layer-by-layer (LBL)process. As shown in FIG. 2A, loaded cationized ferritin cages 230loaded with an oxidizer (iron oxide in this example) are assembled in asingle layer on the surface of a nano-Al particle 250 to form asingle-layer reactive nanocomposite 260 a similar to that shown in FIG.1A. The loaded cationized ferritin cages 230 may have a zeta potentialof approximately +23.6±8.0 mV (obtained on a Malvern Instruments® nanoseries Zetasizer®). The loaded cationized ferritin cages 230 interactstrongly with the surface of the nano-Al particle 250 and provide anabundance of surface charge for assembly with a second protein layercontaining an opposite electrostatic charge. Loaded native (unmodified)ferritin cages 270 loaded with an oxidizer (AP in this example) are thenassembled in a single layer on top of the layer of loaded cationizedferritin cages 230 to form a two-layer reactive nanocomposite 280. Theloaded native ferritin cages 270 are negatively charged and may have azeta potential of approximately −32.9±8.7 mV. A layer of loadedcationized ferritin cages 230 (loaded with iron oxide in this example)may be assembled onto the layer of loaded native ferritin cages 270 toform a three-layer reactive nanocomposite 290. Additional alternatinglayers of (oppositely charged) loaded native ferritin cages 270 andloaded cationized ferritin cages 230 may be added to achieve the desiredcomposition and number of layers.

FIG. 2B depicts an alternative embodiment of a multi-layer hybridreactive nanocomposite comprising two or more layers of different typesof proteins and protein cages achieved by an LBL process. Loadedcationized ferritin cages 240 (loaded with AP in this example) areassembled onto nano-Al particles 250 to form a single-layer reactivenanocomposite 260 b similar to that shown in FIG. 1A. An oxidizer (ironoxide 210 in this example) is coated with a negatively chargedpolyelectrolyte 200 to form a negatively charged polyelectrolyte complex245, which is then assembled on top of the layer of loaded cationizedferritin cages 240 to form a multi-layer hybrid reactive nanocomposite295. Similar to the embodiment depicted in FIG. 2A, additionalalternating layers of loaded cationized ferritin cages 240, loadednative ferritin cages (not shown), and/or negatively chargedpolyelectrolyte complexes 295 may be added to achieve the desiredcomposition and number of layers.

FIGS. 3A-F are TEM micrographs (obtained on a Philips CM200 TEMoperating at 200 kV) of single- and multi-layer reactive nanocompositesrespectively containing between one and six alternating homogeneouslayers of cationized and native ferritin loaded only with iron oxide. Intotal, up to 12 layers of iron oxide loaded ferritin have beensuccessfully loaded onto nano-Al with good coverage (data not shown).

In all embodiments of the reactive nanocomposite according to thepresent invention, the oxidizer may comprise iron oxide, AP, or both. Inone embodiment, the oxidizer comprises all iron oxide. In anotherembodiment, the oxidizer comprises all AP. In further embodiments suchas those depicted in FIGS. 2A and 2B, the oxidizer may comprise ironoxide and AP in alternating layers. The interior protein contents (ironoxide particle or ammonium perchlorate) may also be varied at any layerby sequential addition of AP/dialysis steps as described below inExample 1 or mineralization of the iron oxide inside the protein cage.Different layers of self-assembled proteins containing both iron oxideand AP may further be assembled as heterogeneous layers and/or varied bythe assembly order with respect to the nano-Al surface (i.e. AB-nAl orBA-nAl). In addition, different types of oxidizers may be loaded intothe same protein cage. The oxidizer may be varied from layer to layerand within the same layer to obtain higher iron oxide loading withnano-Al and to achieve the desired stoichiometric conditions and wt %,thereby achieving a reactive nanocomposite having the desiredreactivity.

The following examples and methods are presented as illustrative of thepresent invention or methods of carrying out the invention and are notrestrictive or limiting of the scope of the invention in any manner.

Example 1 Assembly of Single-Layer Ferritin-Nano-Al

2 mg of aluminum nanoparticles (NovaCentrix® Inc., 80 nm, 80% active Alcontent) passivated with an amorphous aluminum oxide is added to 100 μLof cationized ferritin from horse spleen (Sigma®, 48 mg/mL) containing acore of iron oxide. The mixture is dispersed and sonicated in 500 μL ofdeionized water. These components are incubated for 1 hour to promotefunctionalization of the nano-Al with protein cages and then purified toremove excess unbound ferritins by centrifugation at 4000 rpm for 10minutes. The isolated ferritin-nano-Al pellet is redissolved in 500 μLof deionized water to achieve a single-layer ferritin-nano-Al materialsimilar to that depicted in FIG. 1A.

To obtain AP-loaded ferritin-nano-Al, the iron oxide core of cationizedferritin is removed by reductive dissolution with 0.5% mercaptopropionicacid in 0.1 M acetate buffer, pH 4.5, and repeated dialysis using 10 kDaMWCO dialysis tubing (Fisherbrand®). The empty cage is then subsequentlyfilled via successive additions of 0.1 M ammonium perchlorate (Sigma®)in water and multiple dialysis steps to obtain maximal loading. Theresult is a single-layer ferritin-nano-Al material similar to thatdepicted in FIG. 1A. Alternatively, to spectroscopically showincorporation, a rhodamine perchlorate analogue (Exciton®) may be usedin place of or along with the AP during loading of the apoferritincages.

Example 2 LBL Assembly of Multi-Layer Ferritin-Nano-Al

Nano-Al particles are first coated with a single layer of cationizedferritin as described above in Example 1. Following centrifugation toremove excess unbound ferritins and resuspension in deionized water, 100μL of native ferritin containing an iron oxide core from horse spleen(Sigma®, 56 mg/mL) is added to the single-layer ferritin-nano-Al andincubated for 15 minutes, followed by centrifugation at 4000 rpm for 10minutes. The pellet is resuspended in 500 μL of deionized water to yieldtwo layers of protein cages surrounding the nano-Al. This process isrepeated to build additional protein layers on nano-Al as shown in FIG.2A by using alternating layers of cationized and native ferritin (i.e.cationized ferritin for the third layer and native ferritin for thefourth layer), with centrifugation and resuspension steps in betweeneach layer to remove unbound ferritins.

Alternatively, the iron oxide core of the cationized ferritin and/or thenative ferritin in any layer may be replaced with AP as described inExample 1 to create a homogeneous AP-loaded ferritin-nano-Al or aheterogeneous iron oxide-AP complex.

Example 3 Characterization of Ferritin-Nano-Al

Materials and Methods

Energy dispersive X-ray (EDAX) spectroscopy was performed using anintegrated EDAX detector from 0-20 keV at an angle of 15°. X-rayphotoelectron spectroscopy (XPS) measurements were performed using anM-PROBE Surface Science® XPS spectrometer utilizing chargeneutralization. Samples were prepared by drop-casting 10 μL of anaqueous suspension of ferritin-nano-Al onto a polished silicon wafer(Wafer World, Inc.), followed by air drying. Spectra were collected in 1eV steps from 0-1000 eV at a spot size of 800 μm and averaged over 15scans for standard resolution.

Ferritin binding was determined using a Q-Sense® E4 QCM-D system withflow modules. Quartz crystal microbalance (QCM) sensors coated with a100 nm aluminum oxide film (Q-Sense, QSX-309) were cleaned by UV/ozonetreatment (Novascan® PSD Pro Series Digital UV/Ozone system) for 10minutes, immersion in a 2% SDS solution for 30 minutes, thorough rinsingwith deionized water, N₂ drying, and another UV/ozone treatment for 10minutes. After cleaning, sensors were mounted in QCM flow modules.Cationized ferritin (Sigma®) and native ferritin (Sigma®) atconcentrations of 96 μg/mL and 56 μg/mL, respectively, in deionizedwater were flowed across the QCM sensors at 0.17 mL/min and monitoredvs. time at the third overtone frequency for LBL assembly.

Dynamic light scattering (DLS) of multi-layer assemblies was performedon a Malvern Instruments® nano series Zetasizer® after addition of eachprotein cage layer. For the LBL assembled ferritin-nano-Al material, SEMand EDAX maps were obtained on a Philips XL series FEG eSEM operating at10 kV and a working distance of 7.5 mm. For imaging and mapping, 20 μLof ferritin-nano-Al was drop-cast on a silicon wafer and mounted on anSEM puck.

Thermogravimetric analysis (TGA) and differential thermal analysis (DTA)measurements were performed in a TA Instruments® SDT Q 600. Samples (5to 10 mg) were placed into a tared alumina crucible with an emptyalumina crucible serving as the reference. All data was collected indynamic mode under flowing argon (100 mL/min) from room temperature upto 1000° C. at a rate of 5° C./min. Control samples were prepared bymixing AP and nano-Al or iron oxide (Fe₂O₃) nanopowder with nano-Al atthe appropriate stoichiometric ratios.

Combustion experiments were performed by placing approximately 10 mg ofthe respective nanocomposite powder onto a flat substrate in a ventedfragmentation chamber under an air atmosphere. The powders were ignitedby a butane flame from directly below. A NAC® Image Technology Memrecam®GX8 digital high speed video camera, collecting full frame, full colorimages at 5,000 frames per second, was used to record the combustionevents.

Composition and Ferritin Binding

EDAX analysis of nano-Al particles functionalized with iron oxide-loadedcationized ferritin cages yielded a concentration of 18.2 wt % Al and1.7 wt % Fe as measured by EDAX (data not shown). In contrast, theassembly of nano-Al with native ferritin (negatively charged) resultedin only a few protein cages being associated with the nano-Al surface byTEM and no detectable Fe (not shown). XPS measurements of nano-Alparticles functionalized with AP-loaded cationized ferritin cagesconfirmed the presence of 0.4% Cl, 5.2% N, and 9.3 atomic % of Al (datanot shown). This measurement is equivalent to a stoichiometry ofapproximately 440 AP molecules/protein cage and represents a fullyfilled cage.

FIG. 4 is a QCM plot (change in mass vs. time) of reactivenanocomposites comprising between one and six layers of ironoxide-loaded ferritins of equal mass, such as the reactivenanocomposites in FIGS. 3A-3F. The formation of alternating layers ofprotein cages may be seen by the step-wise increase in masscorresponding to each new layer. DLS analysis further confirmed theformation of layers of protein cages, which is reflected in FIG. 5 by anapproximately linear increase in nanoparticle size with the addition ofeach protein layer.

Energetic Characterization of Ferritin-Nano-Al

Initial measurements of the energetic performance of theferritin-nano-Al materials were obtained by simultaneous TGA and DTA.FIG. 6 shows the TGA/DTA profile of bio-thermite (nano-Al functionalizedwith a single layer of iron oxide-loaded cationized ferritin;

). As a baseline, the TGA/DTA profile of unfunctionalized nano-Al (

) particles exhibited a broad exotherm occurring between 100-350° C. dueto the conversion of amorphous-Al₂O₃ to gamma-Al₂O₃ and a sharpendothermic peak at 660° C. from the melting of nano-Al. Thischaracteristic melting peak provides a means to assess how much aluminumis consumed during the course of the reaction and whether or notstoichiometric conditions are reached. For the bio-thermite sample (50wt % Fe:50 wt % nano-Al), the exotherm appeared prior to aluminummelting, with a reaction onset at approximately 300° C. for 1.7 wt %FeO(OH) loaded ferritin. Additionally, a small endothermic peak wasobserved at approximately 800° C., which is also present in the TGAprofile of ferritin alone (not shown). The peak at 800° C. may beattributed to a phase transition of ferrihydrite FeO(OH) to magnetite(Fe₃O₄).

FIG. 7 shows the TGA/DTA profile of AP-loaded ferritin-nano-Al.Energetically, the profile of AP loaded ferritin-nano-Al (

) showed similar exothermic behavior to the bio-thermite sample in FIG.6. However, the entire event for the AP-loaded ferritin-nano-Al wasexothermic due to the complete consumption of nano-Al prior to melting.In this case, the reactants were stoichiometrically balanced using onlya single layer of cationized ferritin molecules filled with AP. TheTGA/DTA profile of unfunctionalized nano-Al (

) is also shown in FIG. 7. By comparison, the addition of bulk APrecrystallized with nano-Al resulted in the thermal decomposition of APbelow 200° C. and unreacted nano-Al by TGA (data not shown). The TGA/DTAprofile of AP-loaded ferritin-nano-Al in FIG. 7 demonstrates the thermalstabilization of AP upon encapsulation by the ferritin protein cage andits critical role in reacting with nano-Al.

FIG. 8 shows the TGA/DTA profiles of multi-layer ferritin-nano-Alcontaining one, two, or four homogeneous layers of iron oxide-loadedferritin, along with the profile for unfunctionalized nano-Al(unfunctionalized nano-Al

; 1-layer iron oxide-loaded-ferritin-nano-Al

; 2-layer iron oxide-loaded-ferritin-nano-Al

; and 4-layer iron oxide-loaded-ferritin-nano-Al

). As compared to one another, the profiles of the multi-layerferritin-nano-Al showed increasing exotherms as the number of layersincreased from one to four, and higher consumption of nano-Al isobserved by a decreased melting nano-Al peak. The exotherm size wasdependent upon the weight ratio of Al to iron oxide, whereby slightlyfuel rich mixtures (50:50) produced the largest exotherm.

For comparison, FIG. 9 shows the TGA/DTA profile for four-layerferritin-nano-Al (

) plotted with the TGA/DTA profile generated from a bulk thermitereaction of micron- or nano-sized iron oxide particles and nano-Alparticles (

). The bulk thermite material consisted of micron size Fe₃O₄ powdermixed with nano-Al powder at 50 wt % Fe:50 wt % nano-Al. In FIG. 9, theexotherm for the bulk thermite reaction was not observed until 750-950°C., and the reaction released a lower amount of heat. In contrast, thebio-thermite reaction yielded larger exotherms at a lower temperaturerange, while also consuming more of the aluminum at near stoichiometricconditions.

Finally, FIG. 10 shows the combustion characteristics of severalreactive nanocomposites utilizing a high speed camera. Unlike TGA/DTA,the burn tests provide a means to qualitatively test the energeticbehavior and performance of each material in a real world scenario andunder normal combustion conditions. The materials were ignited using aflame from a butane torch and recorded by a high speed digital videocamera during the course of combustion, followed by analysis of selectedindividual time frames. A two-layer apoferritin-coated nano-Al sample(no iron oxide core) shown in FIG. 10A serves as a control. Thetwo-layer apoferritin-nano-Al powder burned slowly, yielding only afaint glow. This minimal combustion was limited by the reaction withatmospheric oxygen. However, incorporation of iron oxide into theapoferritin generated a much greater combustion event as seen FIG. 10B,which shows the combustion of a two-layer iron oxide-loaded ferritinnano-Al material. The combustion reaction of the iron oxide-loadedferritin nano-Al material may be as follows: 2nano-Al+3FeO(OH)→Al₂O₃+3Fe+30H⁻. The combustion event in FIG. 10Bfeatured a large intense flame and the appearance of several sparksgenerated due to the flocculant nature of the electrostatically chargedpowder.

A significant increase in the amount of fuel (iron oxide) as in the12-layer iron oxide-loaded ferritin-nano-Al material shown in FIG. 10Cresulted in an even larger flame that burned faster. By comparison, moreiron oxide was consumed in the 12-layer material, leading to fastercombustion and release of more energy. Conversely, upon substitution ofthe iron oxide for the stronger oxidizing agent AP, the single-layerAP-loaded ferritin-nano-Al material in FIG. 10D achieved an impressivecombustion that lasted a much shorter time. The combustion reaction ofthe AP-loaded ferritin-nano-Al material may be as follows: 8nano-Al+3ClO₄→Al₂O₃+3Cl⁻. In FIG. 10D, the nano-Al was rapidly consumedupon reacting with the encapsulated AP, and the reaction was essentiallyfinished after 0.16 sec.

As described above, single- and multi-layer ferritin-nano-Al materialsdemonstrate enhanced reaction rates and increased energy output. Theprotein cages (1) offer the ability to encapsulate and thermallystabilize an inorganic material such as iron oxide or an oxidizing agentsuch as AP; (2) interact with and coat the surface of reactivenanometals such as nano-Al; and (3) quickly deliver the oxidizer to thereactive nanometal surface by reducing the diffusion distance and masstransport of reactants. By varying the number and composition of theprotein layers, the reaction stoichiometry of the nanometal with theoxidizer may be tightly controlled in order to tailor the energeticproperties to the desired application. Potentially, each protein layermay be customized with an inorganic material, oxidizing agent, molecularexplosive, and/or other reactant as desired. For example, as seen inFIGS. 10A-D, the use of iron oxide-loaded ferritin nano-Al led to aslower sustained burn, which may be attractive for propellant orpyrotechnic applications, whereas the fast and intense combustionproduced by the AP-ferritin-nano-Al materials may serve as a usefuladditive for explosive materials. Using a bio-derived route alsoprovides the opportunity for the development of safer and more efficientcombustible materials. For example, free AP is generally sensitive toshock and friction, decomposes at low temperatures, and becomesexplosive when mixed with metals. Encapsulation of AP in a protein cagereduces or prevents these issues, making it safer to handle and process.Furthermore, the protein cages, due to their organic nature, may serveas gasification agents during the reaction. The heat produced during thereaction decomposes the protein cages into various carbonaceous gaseousproducts that will further increase the reaction pressure.

Although this invention has been described with respect to certainpreferred embodiments, various other embodiments and various changes andmodifications to the disclosed embodiment(s) will become apparent tothose skilled in the art. All such other embodiments, changes, andmodifications are intended to come within the spirit and scope of theappended claims.

What is claimed is:
 1. A method of making a reactive nanocomposite,comprising: forming a layer of a plurality of positively-charged loadedprotein cages onto an outer surface of a plurality of metalnanoparticles to form the reactive nanocomposite, wherein the outersurface comprises at least one metal selected from the group consistingof Al, B, Si, Mg, Ni, Ti, and Ag, and wherein the plurality ofpositively-charged loaded protein cages comprise a protein cage having afirst oxidizer loaded into a pore of the protein cage.
 2. The method ofclaim 1, wherein the first oxidizer is selected from the groupconsisting of iron oxide and ammonium perchlorate.
 3. The method ofclaim 1, wherein the positively-charged loaded protein cages comprise aself-assembling protein that is selected from the group consisting ofcationized ferritin, heat shock proteins, capsid proteins, andferritin-like proteins.
 4. The method of claim 1, wherein thepositively-charged loaded protein cages further comprise an additionalreactant selected from the group consisting of a secondary chemicaloxidizing agent, a non-ferric metal oxide, a molecular explosive, and afluorescent dye taggant.
 5. The method of claim 3, wherein theferritin-like proteins are selected from the group consisting of Dpsproteins and Dpr proteins.
 6. The method of claim 1, further comprising:loading a plurality of empty protein cages with the first oxidizer toform the loaded positively-charged protein cages.
 7. A method of makinga multi-layered reactive nanocomposite having a charged outer surface,the method comprising the steps of: a) forming a layer of a plurality ofpositively-charged loaded protein cages onto an outer surface of aplurality of metal nanoparticles to form a reactive nanocomposite havinga positively-charged outer surface; b) forming a layer of a plurality ofi) negatively-charged loaded protein cages, or ii) negatively-chargedloaded polyelectrolyte complexes onto the reactive nanocomposite havingthe positively-charged outer surface to form a multi-layered reactivenanocomposite having a negatively-charged outer surface; and c)optionally repeating step a) to form a multi-layered reactivenanocomposite having a positively-charged outer surface, or optionallyrepeating steps a) and b) to form the multi-layered reactivenanocomposite having the negatively-charged outer surface, wherein theplurality of positively-charged loaded protein cages comprise a firstprotein cage having a first oxidizer loaded into a pore of the firstprotein cage; wherein the plurality of negatively-charged loaded proteincages comprise a second protein cage having a second oxidizer loadedinto a pore of the second protein cage, and wherein the plurality ofnegatively-charged polyelectrolyte complexes comprise a third oxidizercoated with a plurality of negatively-charged polyelectrolytes.
 8. Themethod of claim 7, wherein the outer surface of the plurality of metalnanoparticles comprise at least one metal selected from the groupconsisting of Al, B, Si, Mg, Ni, Ti, and Ag.
 9. The method of claim 7,wherein the first oxidizer the second oxidizer, and the third oxidizerare independently selected from the group consisting of iron oxide andammonium perchlorate.
 10. The method of claim 7, wherein thepositively-charged protein cages comprise a self-assembling protein thatis selected from the group consisting of cationized ferritin, heat shockproteins, capsid proteins, and ferritin-like proteins.
 11. The method ofclaim 10, wherein the ferritin-like proteins are selected from the groupconsisting of Dps proteins and Dpr proteins.
 12. The method of claim 7,wherein the positively-charged protein cages further comprise anadditional reactant selected from the group consisting of a secondarychemical oxidizing agent, a non-ferric metal oxide, a molecularexplosive, and a fluorescent dye taggant.
 13. The method of claim 7,further comprising: coating a surface with the plurality ofnegatively-charged polyelectrolytes to form a coated surface; andassembling the reactive nanocomposite having the positively-chargedouter surface or the multi-layered reactive nanocomposite having thepositively-charged outer surface onto the coated surface with theplurality of negatively-charged polyelectrolytes, thereby directingassembly of the multi-layer reactive nanocomposite onto the surface. 14.The method of claim 7, further comprising: loading a plurality of emptyfirst protein cages with the first oxidizer to form the loadedpositively-charged protein cages; loading a plurality of empty secondprotein cages with the second oxidizer to form the loadednegatively-charged protein cages; or coating the third oxidizer with theplurality of negatively-charged polyelectrolytes to form thenegatively-charged polyelectrolyte complexes.
 15. The method of claim 7,wherein the multi-layered reactive nanocomposite comprises two to twelvelayers of alternating charged layers comprising a) positively-chargedloaded protein cages; and b) i) negatively-charged loaded protein cages,and/or ii) negatively-charged polyelectrolyte complexes to provideeither the multi-layered reactive nanocomposite having thepositively-charged outer surface, or the multi-layered reactivenanocomposite having the negatively-charged outer surface.